The present invention, in some embodiments thereof, relates to bacterial polypeptides that comprise toxin or antitoxin activity and, more particularly, but not exclusively, to toxin-antitoxin pairs that may be used as bacterial anti-phage defense systems.
A broad array of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various substrates. Because enormous amounts of bacteria are being cultivated each day in large fermentation vats, bacteriophage contamination can rapidly bring fermentations to a halt and cause economic setbacks, and is therefore considered a serious threat in these industries. The dairy fermentation industry has openly acknowledged the problem of phage and has been working with academia and starter culture companies to develop defense strategies and systems to curtail the propagation and evolution of phages for decades.
To survive in the face of perpetual phage attacks, bacteria have developed a variety of anti-phage defense systems (Labrie et al., 2010; Stern and Sorek, 2011). These systems include restriction enzymes that recognize and cleave foreign DNA (King and Murray, 1994), abortive infection (Abi) mechanisms that lead the bacterial cell, upon phage invasion, to commit “suicide”, thus protecting the colony against phage spread (Chopin et al., 2005); and the recently identified adaptive defense system called CRISPR/Cas, which uses small RNAs to target invading phage DNA (Deveau et al., 2010; Horvath and Barrangou, 2010; Sorek et al., 2008; van der Oost et al., 2009). Due to the rapid evolution and elaborated biological novelty associated with the bacteria-phage arms race, it is estimated that many additional, yet uncharacterized anti-phage defense systems are encoded by bacteria and archaea to (Makarova et al., 2011; Stern and Sorek, 2011). As part of this continuous arms race, successful phages had also developed numerous counter-resistance mechanisms to overcome bacterial defense (Labrie et al., 2010; Stern and Sorek, 2011).
The growing availability of genomic sequences has elucidated the vast dispersion of toxin-antitoxin (TA) systems in prokaryotic genomes (Shao et al., 2011). These systems (also called TA modules), composed of a toxic gene and a neutralizing gene, were first suggested to function as plasmid ‘addiction molecules’ (Van Melderen and Saavedra De Bast, 2009; Wozniak and Waldor, 2009), but their prevalent existence on chromosomes (Aizenman et al., 1996; Makarova et al., 2009; Shao et al., 2011) has led to the understanding that this is unlikely their major role. Accumulating evidence suggest that TA modules play pivotal roles in prokaryotic cellular biology including programmed cell death (Hazan et al., 2004), stress response (Christensen et al., 2001), generation of persister cells (Schumacher et al., 2009), biofilm formation (Kim et al., 2009) and phage defense via abortive infection (Fineran et al., 2009; Hazan and Engelberg-Kulka, 2004; Koga et al., 2011; Pecota and Wood, 1996).
The most prevalent kind of TA systems is type II systems, where both toxin and antitoxin are proteins (as opposed to types I and III where the antitoxin is a non-coding RNA (Fineran et al., 2009; Fozo et al.)). The two genes, which reside on the same operon, code for small proteins and inhibition of the toxin is carried out through protein-protein interaction. As a rule, the toxin is a stable protein and the antitoxin is unstable and degrades rapidly by one of the housekeeping bacterial proteases, usually Lon or ClpP (Aizenman et al., 1996; Cherny and Gazit, 2004; Christensen et al., 2004; Christensen et al., 2001; Christensen et al., 2003; Lehnherr and Yarmolinsky, 1995; Roberts et al., 1994; Van Melderen et al., 1996). As a result, continuous production of the antitoxin is required to prevent the toxin's deleterious effects (Van Melderen and Saavedra De Bast, 2009).
A number of reports demonstrate a role for type II TA systems in phage resistance via an Abi mechanism: the extensively studied MazEF system was shown to eliminate P1 phage infection (Hazan and Engelberg-Kulka, 2004), and the recently identified RnlA toxin which is activated following T4 phage infection degrades phage mRNAs (Koga et al., 2011). Type I and Type III TA systems (hok/sok and ToxIN, to respectively) were also shown to provide resistance against phages (Fineran et al., 2009; Pecota and Wood, 1996). However, based on their presence in bacterial ‘defense islands’, it was hypothesized that TA systems play a much wider role in phage defense (Makarova et al., 2011).
The usage of Abi systems for biotech purposes, especially in protecting dairy industry bacteria from bacteriophages is disclosed in U.S. Pat. Nos. 7,754,868, 7,550,576, 7,169,911 and 5,994,118.
To date, at least 12 families of type II TA systems have been described (Masuda et al., 2012; Shao et al., 2011). Members of these families are widespread in bacterial and archaeal genomes and undergo extensive horizontal gene transfer (Guglielmini et al., 2008; Makarova et al., 2009; Pandey and Gerdes, 2005; Shao et al., 2011). Computational studies based on the ‘guilt by association’ principle (gene neighbors of known toxins/antitoxins are suspected as antitoxins/toxins themselves) and on other characteristics of TA systems have recently predicted several novel TA families (Leplae et al., 2011; Makarova et al., 2009), which are yet to be validated experimentally.